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Application of gold-silver mixed-metal cluster compound in preparing fluorescence staining reagent for cell nucleolus

A technology for cell nucleolus and fluorescent staining, which is applied in the application field of gold-silver heterometallic clusters in the preparation of cell nucleolus fluorescent staining reagents, and can solve the problems of free radical cell sample damage, cells prone to light damage, and poor photostability , to achieve strong anti-light quenching ability, long fluorescent signal labeling time, and stable imaging

Inactive Publication Date: 2012-11-28
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current fluorescent labeling probes for cells have the following disadvantages: First, the photostability is poor, and photofloating occurs rapidly under the continuous irradiation of excitation light.
Second, cells are prone to photodamage, especially in the presence of fluorophores, and free radicals generated during photobleaching can damage cell samples

Method used

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  • Application of gold-silver mixed-metal cluster compound in preparing fluorescence staining reagent for cell nucleolus
  • Application of gold-silver mixed-metal cluster compound in preparing fluorescence staining reagent for cell nucleolus
  • Application of gold-silver mixed-metal cluster compound in preparing fluorescence staining reagent for cell nucleolus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Nucleolar fluorescent staining of gold-silver heterometallic clusters:

[0017] 1) Human cervical cancer cell line HeLa (purchased from Shanghai Cell Bank, Chinese Academy of Sciences), in DMEM medium containing 10% fetal bovine serum, 37°C, 5% CO 2 , cultivated under saturated humidity conditions. Replace the culture medium every two days, and subculture every 3 to 4 days.

[0018] 2) Handling of cell culture slides. Wash the Φ14mm coverslip with water, soak in sulfuric acid-potassium dichromate solution for 24 hours, wash with distilled water, soak in absolute ethanol, and set aside.

[0019] 3) Preparation of cell slides. Spread the coverslips in a Φ35mm Petri dish in a 1×10 5 Cells / mL seeded, 37°C, 5% CO 2 , placed in an incubator for cultivation.

[0020] 4) After the cells adhere to the wall, wash with phosphate buffered solution (PBS) 3 times, add a gold-silver heterometallic cluster solution with a final concentration of 10 μM and incubate at room temperat...

Embodiment 2

[0024] Colocalization fluorescent staining: see Example 1 for specific steps. After staining with gold and silver heterometallic clusters, wash with PBS and add 2 mL of 4% paraformaldehyde fixative, fix for 30 min, and add commercial nucleolus staining reagent Syto Green RNA Select (Invitrogen) was further incubated for 20 min, and then the solution was aspirated, and the cells were washed 3 times with PBS, each time for 3-5 min. Fluorescent signals were collected from cells incubated with clusters and Syto Green RNA Select with 405nm and 488nm lasers respectively (gold and silver heterometallic clusters: 570±20nm; Syto Green RNA Select: 530±20nm)

[0025] Results: For details, see image 3 and 4 , it can be observed from the figure that the cell nucleolus marked by the cluster compound of the present invention can be well matched with the commercialized cell nucleolus staining reagent Syto Green RNA Select, thus proving that the labeling method of the present invention can c...

Embodiment 3

[0027] Intracellular photostability of clusters and DAPI: see Example 1 for specific steps. After staining with clusters, wash with PBS and add 2 mL of 4% paraformaldehyde fixative, fix for 30 min, add organic dye DAPI for further incubation for 20 min, and then Aspirate the solution, and wash the cells 3 times with PBS, each time for 3-5 minutes. Cells were continuously irradiated with a 405nm laser for time-series scanning.

[0028] Results: For details, see Figure 5 and 6 , it can be observed from the figure that the cell nucleolus region marked by the method of the present invention shows a strong fluorescent signal, and as time goes by (420s), the photostability of the cluster is higher than that of the organic dye DAPI, thus proving that , the labeling method of the present invention has high fluorescence stability.

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Abstract

Application of gold-silver mixed-metal cluster compound in preparing fluorescence staining reagents for cell nucleoli relates to a fluorescence staining reagent. The invention provides an application of the gold-silver mixed-metal cluster compound which has excellent specific staining effect to cell nucleolus area quickly and stably, in preparing fluorescence staining reagents. The gold-silver mixed-metal cluster compound [Au6Ag2(C)(dppy)6](BF4)4 is synthesized using the method in the document: Jian-Hua Jia, Quan-Ming Wang; J. Am. Chem. Soc. 2009, 131, 16634-16635. The dppy is diphenyl-2-pyridylphosphine. The gold-silver mixed-metal cluster compound has fluorescence staining effect to cell nucleolus area, wherein the cell nucleolus fluorescence staining performance of the gold-silver mixed-metal cluster compound reaches or exceeds that of existing like products, so the compound can be used for preparing fluorescence staining reagents for cell nucleoli.

Description

technical field [0001] The invention relates to a fluorescent dyeing reagent, in particular to the application of a gold-silver heterometallic cluster in the preparation of a cell nucleolus fluorescent dyeing reagent. Background technique [0002] The nucleolus is the most prominent structure in the interphase nucleus of eukaryotic cells, which is a single or multiple homogeneous spherical bodies. The size, shape and number of nucleoli vary with the species of organism, cell type and cellular metabolic state. The nucleolus in eukaryotic cells has important functions as the site of rRNA synthesis, processing and assembly of ribosomal subunits. Therefore, the research on the structure, dynamics and function of the nucleolus has not only been paid close attention to by early cytologists, but also has been highly valued by researchers in various related fields since the discovery of the important functions of the nucleolus in the 1960s. The earliest observation of the structur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30
Inventor 王泉明李富友雷振陈敏
Owner XIAMEN UNIV
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